1. Field of the Invention
The present invention relates to an automated apparatus and method for attaching a labeled probe and/or an antibody to macromolecules such as nucleic acid fragments and proteins.
2. Description of the Prior Art
The detection of certain macromolecules such as nucleic acid ("DNA/RNA") strands or proteins of interest which have been physically separated from other macromolecules on a molecular weight basis, for example, by electrophoresis and transferred to a filter or membrane has generally been accomplished manually. Typically the membrane carrying the separated macromolecules is inserted in a plastic bag (or box).
A plurality of reagents are sequentially added to and removed from the bag with the bag being sealed after each reagent is added so that the reagent can be agitated, for example, by shaking the bag to uniformly dispose the membrane to the reagent. The bag then must be unsealed when one reagent is to be removed and a new one added.
As an example, the Biochemicals Division of Boehringer Mannhein of Indianapolis, Ind. ("Boehringer") suggests the following procedure for hybridizing labeled (non-radioactive) DNA to immobilized target DNA affixed to a nitrocellulos filter or nylon membrane. The filter or membrane is initially prehybridized by sealing each filter in a plastic bag (or box) with a quantity (e.g. 20 ml/100 cm.sup.2 of filter) of hybridization solution for about one hour. The solution is redistributed over the filter periodically by manually moving or shaking the bag.
The bag is then opened and the prehybridization solution removed and replaced with an additional quantity of hybridization solution containing a small quantity (e.g., 25 ng) of labeled and freshly denatured DNA. Care must be taken to prevent the filters from becoming dry when exchanging the solutions.
The bag is then resealed and the filter incubated at an appropriate temperature for several hours. To ensure the exposure of the filter to the labeled DNA and hybridization solution during the incubation period, the bag should be shaken or otherwise agitated from time to time.
The bag is them reopened, the excess labeled DNA and solution removed. A wash solution is then inserted into the bag, the bag sealed and agitated to further remove unattached labeled DNA and hybridization solution from the bag. Additional washing steps may be used.
An antibody conjugate solution replaces the wash solution and the filter is incubated for an appropriate time to allow the antibody to bind to the labeled DNA. The unbound antibody is removed and the filter washed. The bag is also agitated during these additional steps. The filter is then exposed to a prepared color developer which reacts with the antibody to produce a colored precipitate identifying the target DNA.
The use of a radioactive substance to form the labeled DNA eliminates the antibody and color forming steps. The location of the radioactive material may be detected by photographic techniques. However, the precautions required in handling radioactive material may more than offset the antibody and color developer steps.
The identity of target proteins separated by, for example, the Western Blotting technique requires steps similar to the steps outlined above with respect to hybridizing labeled non-radioactive DNA to target DNA except that the labeled probe consists of an antibody which contains a phosphorescent material or has an enzyme attached to it which will allow for either a color substrate development or a chemiluminescent development, as is well known to those skilled in the art.
All such macromolecule identification techniques require that the filter or membrane be exposed sequentially to several reagents. The manual operation required to fill, seal, open, drain and refill the bag for each step is time consuming and costly. In addition one or more of the reagents such as the labeled probe (DNA/RNA strand or proteins) and the antibody conjugate (for non-radioactive DNA/RNA detection) are very expensive. To conserve costs it is desirable to use very small quantities of such reagents. However, the ability to ensure that the filter is uniformly exposed to the reagents during the incubation step by manually agitating or shaking the bag is not compatible with a very small volume of solution.
There is a need for a more efficient and less costly system for identifying macromolecules of interest which have been separated from other macromolecules and bound to a filter or membrane.